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The Journal of Neuroscience, July 1, 1999, 19(13):5666-5673
An Antisense Oligonucleotide Reverses the Footshock-Induced
Expression of Fos in the Rat Medial Prefrontal Cortex and the
Subsequent Expression of Conditioned Fear-Induced Immobility
Bret A.
Morrow2,
John
D.
Elsworth1, 2,
Fiona M.
Inglis2, and
Robert H.
Roth1, 2
Laboratory of Neuropsychopharmacology, Departments of
1 Pharmacology and 2 Psychiatry, Yale
University School of Medicine, New Haven, Connecticut 06520-8066
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ABSTRACT |
The immediate-early genes, including c-fos, have
been proposed to be involved in learning and memory. In this report, we
examine stress-induced Fos-like immunoreactivity (Fos-li) in subregions of the prefrontal cortex during a conditioned fear paradigm. During the
acquisition phase, the rats were conditioned to fear a formerly neutral
tone by pairing the tone with a mild footshock. The rats were then
tested for fearful behavior by reexposure to the tone without
additional footshock. During acquisition, Fos-li was increased in the
medial prefrontal cortex (infralimbic and prelimbic) but not the
anterior cingulate and M1 motor cortex. However, during the extinction
phase, no significant increase in Fos-li was observed in any region.
These findings indicate that acquisition, but not extinction, of
conditioned fear is associated with an increase in Fos-li in subregions
of the medial prefrontal cortex. In other animals, an antisense
oligonucleotide directed against the c-fos mRNA was injected into the
infralimbic/prelimbic cortex 12 or 72 hr before the acquisition
session. Antisense treatment given 12, but not 72, hr earlier
suppressed Fos production without altering behavior during the
acquisition session. Three days after the acquisition session, rats
were tested for fearful behavior as before. The antisense
oligonucleotide blockade of Fos production during acquisition was
associated with a significantly less fearful response during the
extinction session. These results support a role for Fos in the medial
prefrontal cortex during the acquisition of aversive learning.
Key words:
immediate-early gene; learning; AP-1; c-fos; infralimbic
cortex; prelimbic cortex
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INTRODUCTION |
c-fos is an oncogene in
the early-immediate gene family encoding for the Fos protein, which is
capable of forming heterodimers with protein products of the
c-jun transcription family. These dimers bind to the AP-1
site on DNA and regulate the transcription of other genes (Morgan and
Curran, 1991 ; Hoffman et al., 1992). The activation of the
immediate-early gene products appears to be a mechanism by which brief
stimuli can trigger long-term changes in genetic expression and, thus,
alter the neuronal response to subsequent events. The expression of the
oncogene c-fos has been noted to increase in rat brain
tissue in response to different stressors, including restraint (Deutch
et al., 1991; Schreiber et al., 1991; Melia et al., 1994), footshock
(Smith et al., 1992), and several animal behavioral models of anxiety
(Duncan et al., 1996). Other stimuli that were not deliberately
stressful can result in increased production of Fos, including isotonic
saline injections (Sharp et al., 1991; Asanuma and Ogawa, 1994),
handling (Campeau et al., 1991), cocaine administration (Graybiel et
al., 1990; Young et al., 1991), and brushing vibrisea (Mack and Mack, 1992).
Because immediate-early genes have been proposed to be involved with
long-term modifications in neuronal biochemistry or structure thought
to underlie learning (for review, see Dragunow, 1996), we have examined
to use Fos activation in the acquisition and the extinction of
conditioned fear, a model of aversive learning. The conditioned fear
protocol consisted of an acquisition session in which the subject is
taught to fear a tone by pairing it with a brief footshock and an
extinction session in which the conditioned fearfulness is tested by
exposing the subject to the tone without footshock. Our expectation was
that Fos-like immunoreactivity (Fos-li) may be helpful in identifying
prefrontal cortical subregions in which potential cellular changes
involved in learning an aversive behavioral response may occur. The
medial prefrontal cortex (mPFC) was selected as a target because of its
involvement in the biochemical response to stress (for review, see
Deutch and Roth, 1990) and its proposed role in cognition and
higher memory functions (Goldman-Rakic, 1987; Posner, 1994). In a
second issue, the role of the stress activation of Fos in the mPFC was
investigated by suppressing the expression of the Fos protein using a
DNA antisense oligonucleotide (ASO) that is complementary to the Fos
mRNA. Previously, this technique has been used successfully to suppress
Fos production (Chiasson et al., 1992; Heilig et al., 1993;
Hooper et al., 1994; Moller et al., 1994; Hunter et al., 1995). In this
way, the role of the stress-induced activation of Fos during the
acquisition of an aversive memory can be tested.
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MATERIALS AND METHODS |
Animals. Adult male Sprague Dawley rats (Camm
Laboratories, Wayne, NJ) weighing between 200 and 250 gm were used in
all experiments. Rats were housed two per cage on a 12 hr light/dark
schedule (8:00 A.M. lights on). Food and water were supplied ad
libitum in the home cage. Rats were brought into the facility at
least 7 d before the start of the experiment to allow adaptation
after traveling to the facility.
Apparatus. The testing apparatus was a Plexiglas and
stainless steel box (24 × 30 × 27 cm) with a stainless
steel bar floor contained in a isolation chamber to minimize external
influences. The cages were lit indirectly by a dim red light. A white
noise generator provided a constant background noise, and the cages were cleaned and dried before each session with 70% ethanol to minimize olfactory cues. The bar floor was wired for scrambled footshock, and the intensity was calibrated to 0.8 mA (Morrow et al.,
1996). The conditioned stimulus was a 2.8 kHz tone lasting 5 sec. The
tone itself was ~5 dB above background noise and alone does not
visibly startle the rats. The unconditioned stimulus was a 0.5 sec
scrambled footshock timed to terminate at the same time as the tone.
Ten trials per each 30 min session were used, with the intertrial
intervals randomly selected by a personal computer to be between 1 and
4 min.
Experimental protocol. All rats were placed into the testing
apparatus for three 30 min daily sessions to allow adaptation of
novelty-induced activation of Fos. From these rats, three groups were
randomly selected to represent the acquisition and extinction sessions
of fear conditioning, as well as a control group (Table 1). Control rats (n = 6)
were placed into the apparatus for two 30 min additional daily
sessions. The first session was without any footshock or tones, whereas
in the second session, 10 tones without footshock were given. Rats in
the acquisition group (n = 7) were given 1 session
without tones or shock, followed the next day by the acquisition
session of a conditioned fear paradigm: 10 tone with footshock
pairings over a 30 min period. The rats in the extinction group
(n = 6) were subjected to the acquisition session (10 tones paired with footshock), followed the next day by the extinction
session (10 tones without footshock). Previous exposure to this
acquisition paradigm resulted in behavioral and biochemical measures of
fear during the extinction session (Morrow et al., 1996). The
additional habituation sessions in the testing apparatus given to rats
in the control and acquisition groups were used to allow the same
number of sessions for rats in all three groups and thus minimize any
possible effect of differential handling. The rats were videotaped for
later analysis of behavior during the last two sessions. At the end of
the final session, all rats were returned to their home cages and,
after 2 hr, were given pentobarbital (Sigma, St. Louis, MO), 65 mg/kg
intraperitoneally, to induce deep anesthesia. These rats were then
transcardially perfused with heparin (1 U/ml) in saline (0.9%; 50 ml),
followed by 0.1 M phosphate buffered paraformaldehyde (4%;
250 ml, pH 7.4). The brains were removed and stored overnight in
phosphate buffered paraformaldehyde at 4°C.
Separate groups of rats were treated with sense (n = 3 each for 12 and 72 hr delays) or antisense (n = 5 or 6 for 12 and 72 hr delays, respectively) oligonucleotides ~12 or 72 hr
before the acquisition session. Control rats received injections of the PBS vehicle (n = 3 each for 12 and 72 hr delays). Rats
were anesthetized using halothane (Halocarbon Laboratory, River Edge,
NJ) and placed into a stereotaxic instrument (David Kopf, Tujunga. CA)
with a heated platform. The skin in the surgical area was shaved,
scrubbed with iodine surgical scrub (Schein, Port Washington, NY), and cut, and the top of the skull was exposed and verified to be in the
flat skull position. Two small hole were drilled above the mPFC and
2 × 30 ga stainless steel injection cannulas were lowered 3 mm
into the mPFC (3.0 mm anterior and 0.75 mm bilateral to bregma). Phosphorothiolated 15-mer sense and antisense oligonucleotides (antisense; 5' GAA-CAT-CAT-GGT-CGT-3') were obtained from
Oligonucleotide Synthesis facility at Yale University School of
Medicine (New Haven, CT), purified by ethanol precipitation,
resuspended in PBS (pH 7.4), and quantitated by absorbance
spectroscopy. Oligonucleotides (5 nmol in 1 µl) or vehicle were
injected using a syringe pump (Harvard Apparatus, Natick, MA) at 0.2 µl/min over 5 min. The ASO sequence, dosage, and presumed duration of
action were based on published reports (Chiasson et al., 1992; Hooper
et al., 1994). The cannulas were left in place for 5 additional minutes
to avoid drawing the oligonucleotide back up the cannula tract. The
holes in the skull were sealed with bone wax (Schein), and the
incision was area was covered with 5% xylocaine ointment (Schein) and
a topical antibiotic ointment (Tri-thalmic; Schein) and sealed with wound clips. After either a 12 or 72 hr delay, animals were conditioned to fear the tone as by exposure to the acquisition session: 10 tones
paired with footshock. Rats were killed 2 hr after the acquisition session and perfused for immunocytochemical analysis as described previously.
In a final experiment, the effect of ASO blocking the
acquisition-induced activation of Fos in the prelimbic (PL)/infralimbic (IL) area on subsequent fearful behavior observed during the
extinction session was tested. Rats were treated with sense
(n = 6) or antisense (n = 7)
oligonucleotides and, after a 12 hr delay, subjected to the acquisition
session. Rats were returned to the home cage for 72 hr and finally
tested with an extended extinction session; 20 tones, without
footshock, were given over a 1 hr period using the stimuli duration and
intertrial interval described previously. The behavior of the animal in
response to the conditioned tones was videotaped and assessed by a
blinded observer. After the completion of the experiment, rats were
killed for histological examination of the injection site using
50 µm sections stained with cresyl violet (Morrow et al.,
1993). Because no Fos activity was expected during the
extinction session (see Results), no sections were stained for Fos.
Immunocytochemistry. After storing overnight in
paraformaldehyde, the brains were washed in 1.0 M cold
phosphate buffer (PB), pH 7.4, cut on a vibratome into 50 µm
sections, and stained using standard immunocytochemistry techniques
(Leranth and Nitsch, 1994). Some sections were cyropreserved for later
analysis. The sections were washed in PB, treated for 10 min in 1%
sodium borohydride to remove unbound aldehydes and repeatedly rinsed in
PB. The sections were then washed briefly with a PB plus 0.3% Triton
X-100 solution and incubated overnight in a rabbit anti-Fos primary
antiserum (Ab-2; 1:400 dilution in 0.1 M PB plus 0.3%
Triton X-100 plus 0.1% sodium azide; Oncogene Science, Cambridge, MA).
The sections were rinsed in PB plus 0.3% Triton X-100 and further
prepared according to the avidin-biotin complex (ABC) technique (Hsu
et al., 1981) using a Vectastain Elite kit (Vector Laboratories, Burlingame, CA) as follows: (1) incubation for 2 hr with a biotinylated goat anti-rabbit secondary antibody (1:250 dilution in PB plus 0.3%
Triton X-100); (2) rinsed with PB (three times for 10 min); and (3)
incubated in an ABC kit for 2 hr at room temperature. The sections were
then washed, and the tissue-bound peroxidase was visualized using a
Ni-intensified diaminobenzidine (DAB) reaction (15 mg of DAB, 40 ml of
PB, 12 mg of NH4Cl, 0.12 mg of glucose oxidase, 600 µl of
0.05 M nickel ammonium sulfate solution, pH 6.0, and 600 µl of 10%  -D-glucose). Finally, the reaction was terminated by repeated washing with PB, and the sections were mounted
on gelatin-coated slides, dehydrated, and covered with coverslips.
Analysis of data. The behavior of the rats during the
acquisition and extinction sessions was videotaped for later
evaluation. Three major measures of behavior were obtained: (1) fearful
behavior associated with the conditioned (tone) or unconditioned
(tone-footshock) stimuli; (2) fearful behavior associated with the
context (handling, chamber, investigator, etc.); and 3) nonfearful
behaviors, including gross exploratory locomotion, vertical locomotion
(rearing), and grooming. Tone-specific fearful behavior during the
acquisition and extinction sessions was measured by immobility
associated with the presentation of the tone, as described previously
(Morrow et al., 1996). Briefly, immobility, no visible movement except those necessary for respiration, was scored by an experimenter blinded
to each subject's treatment during a 1 min interval after the start of
each of the tones. For context-based fearful behavior, the immobility
of the rat was measured at the beginning of the extinction session over
a 1 min interval after first reexposure to the shock chamber and before
any tones had sounded. No tones were given until after this measure had
been made. Nonfearful behavioral measures were made over a 20 min
interval starting after a 5 min adaptation period in the extinction
session. This interval included tones. During viewing of the videotaped
behavior, the chamber was divided into four equal quadrants by two
lines drawn on the screen. Horizontal locomotion was measured by
counting the number of times the base of the tail crossed one of these lines. The time spent grooming and time spent rearing up with the front
legs off the floor of the cage (vertical locomotion) were also counted.
All statistics were performed on a Macintosh computer using SuperANOVA
version 1.11 (Abacus Concepts, Berkeley, CA). Repeated measures ANOVA
was used for the behavioral data from the acquisition and extinction
sessions with Greenhouse-Geisser correction for covariance (Vitaliano
et al., 1981). Univariate analysis of individual time points within the
acquisition and extinction sessions were done using one-way ANOVA. In
both tests, post hoc testing was performed using the
Newman-Keuls range test.
For biochemical measures of Fos activity, Fos-li nuclei were counted in
representative areas (0.31 mm2) (Fig.
1) of four subdivisions of the PFC [the
IL, PL, anterior cingulate cortex (aCg), and M1 motor cortex (M1)]
using a microscope (Olympus BH-2; Olympus Optical, Tokyo, Japan), a CCD
camera-frame grabber board (Scion Corp., Frederick, MD), and a
MacIntosh computer running the public domain NIH Image (developed at
the National Institutes of Health and available on the Internet at
http://rsb.info.nih.gov/nih-image). Every fifth section was analyzed
between 2.2 and 3.2 mm anterior to bregma according to Paxinos and
Watson (1997) for a total of five sections per animal. The
immunocytochemical data from one animal in the acquisition group was
lost as a result of technical problems. Initially, one section
was photographed and counted visually by three observers (intrarater
reliability, r > 0.95). These preliminary results
using manual techniques were similar to those obtained using the CCD
camera-frame grabber board. To determine the number of Fos-li nuclei
in tissue from rats given local injections into the PL/IL, the stained
sections that included the lowest point of injection were drawn on a
camera lucida, and a 1 mm2 box was drawn around each
injection site, as well as the square millimeter above and
below. The number of Fos-li nuclei in each square millimeter box were
counted by eye instead of using the CCD camera because the cannula
tract interfered with automated counting. In two subjects, it was not
possible to count Fos-li above the injection site because of the damage
from the injection cannula. Repeated measures ANOVA with Newman-Keuls
range test was used to determine group differences. Univariate analysis
of individual subregions of the PFC were done using one-way ANOVA and
Newman-Keuls range test. p < 0.05 was considered
significant.
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Figure 1.
Representation of subregions of the medial
prefrontal cortex and the approximate site of the photomicrograph
taken. The drawing is from Paxinos and Watson (1997) and represents 2.7 mm anterior to bregma.
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RESULTS |
Fos immunocytochemistry and behavior during a conditioned
fear protocol
As observed previously (Morrow et al., 1995, 1996, 1997), the
behavioral paradigm used in this study rendered rats fearful of a
formally neutral tone. For analysis, the behavioral data of all
footshocked rats were used (those being killed for biochemical measurement of Fos-li immediately after the acquisition session, as
well as those conditioned fear rats that went on to the extinction session). During acquisition, rats exposed to tones paired with footshock were significantly more immobile than nonshocked rats exposed
to the tones alone (acquisition, treatment,
F(1,16) = 51.7; p < 0.0001; time, F(9,144) = 6.3;
p < 0.0001; and interaction, F(9,144) = 4.5; p = 0.007)
(Fig. 2). A closer examination revealed that rats receiving footshock remained significantly more immobile than
the nonshocked controls at every time point examined. During subsequent
exposure to the conditioned tone alone, rats previously exposed to the
tone and footshock pairings remained immobile (expression, treatment,
F(1,8) = 170.7; p < 0.0001; time, F(9,72) = 4.5;
p < 0.0001; and interaction,
F(9,72) = 6.2; p = 0.004)
(Fig. 2). A closer examination revealed that conditioned rats were
significantly more immobile after all but the last two
tones.
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Figure 2.
Immobility in rats during the acquisition and
expression phases of the conditioned fear paradigm. After 3 d of
habituation to the testing chamber, rats were subjected to a 2 d
paradigm. On day 1 (top), rats were placed into a
footshock apparatus and randomly received 10 5 sec tones paired
with 0.5 sec footshocks over a 30 min period. Control rats received no
footshocks. Some controls and footshocked rats were killed after the
day 1 session for biochemical analysis. On day 2 (bottom), the remaining rats were returned to the
chamber and exposed to 10 5 sec tones without footshock over a 30 min
period. Seconds spent immobile during 1 min intervals starting with
each of the 10 tones was measured. *p < 0.05 versus control.
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The number of Fos-li nuclei in the IL, PL, aCg, and M1 subdivisions of
the prefrontal cortex were analyzed using a CCD camera-image acquisition system. Representative photomicrographs of the PL region
from rats undergoing the control, acquisition (footshock), and
extinction sessions are shown in Figure
3. Analysis of the number of Fos-li
nuclei in each region indicated a significant difference with regard to
treatment (F(2,15) = 42.2;
p < 0.0001), region
(F(3,45) = 69.4; p < 0.0001), and interaction (F(6,45) = 24.1;
p < 0.0001). A closer analysis revealed higher levels of Fos-li nuclei in rats undergoing the acquisition session (tones with
footshocks) compared with the controls and those undergoing the
extinction session (tones) in the IL
(F(2,15) = 26.4; p < 0.0001) and PL (F(2,15) = 35.6;
p < 0.0001) but not the aCg
(F(2,15) = 3.2; p = 0.07)
or M1 (F(2,15) = 0.4; p = 0.65) (Fig. 4). Rats undergoing the
extinction day of the conditioned fear paradigm had significantly
more Fos-li than the controls but less than rats undergoing the
acquisition session in the PL and IL (p > 0.05).
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Figure 3.
Photomicrographs of the prelimbic region from
representative control, acquisition (footshock), and expression
animals. Rats previously adapted to the testing chamber underwent a
2 d test session. On day 1, rats were placed into the chamber for
30 min and received randomized 10 tones alone
(Control) or paired with footshock
(Acquisition). After this first session, all
rats were returned to the home cages, and, after 90 min, rats in the
acquisition group and some control rats were killed, and the brain
tissues were prepared to visualize Fos-li. On the second day, the
remaining rats were returned to the test chamber and exposed to 10 tones without footshock. Rats previously exposed to footshock on day 1 made up the conditioned fear group (Extinction). Ninety
minutes after the end of the session, these rats were killed, and
Fos-li was visualized in brain tissues. Scale bar, 100 µm.
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Figure 4.
The density of Fos-li-positive nuclei in the
subregions of the mPFC of rats undergoing the acquisition (day 1) or
expression (day 2) of conditioned fear. Fos-li immunocytochemistry was
performed on the brains of rats subjected to the paradigm described in
the legend of Figure 2. *p < 0.05 versus the
control group;  p < 0.05 versus the extinction
group of the same region.
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Antisense effects on Fos biochemistry and behavior
The site of the injection was centered on the lower PL and upper
IL cortex region (Figs. 5,
6) with no points of administration located in the aCg subregion. Antisense injections 12, but not 72, hr
before the acquisition session significantly diminished footshock-induced extinction of Fos-li in the square millimeter surrounding the injection site (Fig.
7, Middle,
F(5,17) = 6.07; p = 0.002).
The effect was limited to the area immediately surrounding the
injections site and did not spread to the square millimeter areas below
or above the injection site area (Fig. 7, Upper,
F(5,17) = 1.73; p = 0.18;
Lower, F(5,17) = 0.44;
p = 0.81). Sense oligonucleotides did not alter the
Fos-li at any time or in any region surrounding the injection site. The
effects of the ASO were gone by 72 hr, and no long-term changes in
footshock-induced Fos activation were observed.
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Figure 5.
The location of the tip of the injection cannula
in rats treated with sense or antisense oligonucleotides in the
prelimbic/infralimbic subregion of the medial prefrontal cortex. Three
groups of animals are represented here. A, Rats treated
12 hr before the acquisition session and killed for
immunocytochemistry. B, Rats treated 72 hr before the
acquisition session and killed for immunocytochemistry.
C, Rats treated 12 hr before acquisition and tested for
expression 72 hr later. The brain drawings are from Paxinos and Watson
(1997), and the numbers represent the location of the
section in millimeters anterior to bregma.
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Figure 6.
A photomicrograph of a representative
injection site from rats injected with either sense oligonucleotide
(SO) and ASO. Rats were treated with the
oligonucleotides (5 nmol in 1 µl) in PBS 12 hr before undergoing the
acquisition session (Fig. 2) and killed 90 min after the end of the 30 min session. Brains were prepared for visualization of Fos-li. Scale
bar, 100 µm.
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Figure 7.
The density of Fos-li positive nuclei in the
square millimeter area surrounding (Middle), above, or
below the injection site of PBS, sense oligonucleotide
(SO), or ASO. Brain sections from treat rats that
underwent the acquisition session of fear conditioning were visualized
for Fos-li and drawn on a camera lucida system. The number of Fos-li
nuclei were counted in each square millimeter area.
*p < 0.05 versus the PBS control group within the
same area.
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The administration of ASO did not alter the acquisition of
footshock-induced immobility in rats treated either 12 or 72 hr earlier (treatment, F(3,34) = 0.65;
p = 0.59; interaction,
F(27,306) = 1.2; p = 0.19) (Fig. 8). There was, however, a
nonsignificant trend for an increase in footshock-induced immobility
during the first footshock in control and ASO rats operated on just 12 hr before compared with those operated on 72 hr before. This is likely a result of the difference in delay after surgery affecting the initial
reactivity of the rat. Unlike during the acquisition session, previous
treatment with ASO 12 hr before the acquisition session resulted in
diminished fear-induced immobility during the expression session
(treatment, F(1,11) = 26.5;
p = 0.0003; time, F(19,209) = 4.7; p < 0.0001; interaction,
F(19,209) = 0.9; p = 0.62)
(Fig. 9). Changes in contextual-based
fear-conditioned immobility were not significantly different
(F(1,11) = 1.23; p = 0.29)
(Table 2). Differences in measures of
nonfear-related behaviors were noted. Treatment with ASO 12 hr
previously increased the measures of horizontal exploratory
locomotion (line crossings) and vertical locomotion (rearing) but not
grooming during the extinction session (line crossings,
F(1,11) = 11.4; p = 0.006;
rearing, F(1,11) = 5.51; p = 0.039; grooming, F(1,11) = 1.06;
p = 0.32) (Table 2).
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Figure 8.
The lack of an effect of antisense oligonucleotide
treatment, given either 12 or 72 hr before, on behavior during the
acquisition session. Rats were treated with sense oligonucleotides
(SO), ASO, or vehicle and, after a 12 or 72 hr delay,
subjected to the acquisition session of the conditioned fear protocol
(Fig. 2). The behavior of the sense oligonucleotide-treated and
vehicle-treated controls were not different and were combined into a
single control group for graphic purposes only. Immobility was measured
for each tone plus footshock pairing over a 1 min interval. There was
no difference between the sense oligonucleotide- or ASO-treated groups
at either 12 or 72 hr.
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Figure 9.
The effect of antisense oligonucleotide blockade
of acquisition-induced Fos-li on the later expression of conditioned
fear. Rats were treated with either sense (SO) or
antisense (ASO) oligonucleotides and, after 12 hr, subjected to the
acquisition session of conditioned fear (Fig. 2). Three days later,
~84 hr after oligonucleotide treatment, rats underwent the expression
session of the conditioned fear protocol. ASO-treated rats showed
significantly less fear-induced immobility than the sense
oligonucleotide-treated controls at every time point tested.
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DISCUSSION |
In this communication, we report two main findings. (1) Using a
mild aversive conditioning protocol, Fos protein was more strongly
activated in association with the acquisition of fear than with the
expression of fear. (2) Blocking the acquisition-induced Fos production
in the PL and IL cortex was associated with a dramatic reduction in the
expression of fear-related behaviors at a time when the ASO was no
longer active in the brain.
Previous studies have proposed a role for Fos and other immediate-early
genes in the process of learning (for review, see Dragunow,
1996). However, some questions have been raised as to the contribution
of novelty and stress to the Fos activation during the learning task
(Asanuma and Ogawa, 1994; Dragunow, 1996). The first of these concerns
was addressed in this current report by habituating the subjects to the
testing environment, thus suppressing novelty-induced Fos activation.
The second point of concern was that stress, independent of any
learning paradigm, has been noted to increase Fos activation (Deutch et
al., 1991; Schreiber et al., 1991; Smith et al., 1992; Melia et al.,
1994; Duncan et al., 1996). The Fos activation during the acquisition
session, however, does not appear to be simply caused by a nonspecific
stress response, because a smaller increase of Fos activation was noted
in the PL/IL during the extinction phase, an interval with robust
changes in behavioral and biochemical correlates of stress (Morrow et al., 1995, 1996, 1997). This suggests that, in this study, Fos activation in the mPFC is not simply related to stress or novelty but
is associated with the acquisition or learning of this aversive conditioning paradigm. Several laboratories, however, have demonstrated increased Fos in regions other than the mPFC during the extinction period of a conditioned stressor (Campeau et al., 1991; Smith et al.,
1992; Beck and Fibiger, 1995). Beck and Fibiger (1995) have shown a
conditioned fear-induced increase in Fos-li in the aCg using a more
severe shock paradigm (150 1 sec footshocks of unknown intensity
per 30 min session for three sessions). Whereas the acquisition period
is believed to reflect learning, the extinction period is thought to
reflect the development of inhibition rather than the deletion of the
acquired memory (Pavlov, 1927; Konorski, 1967; Bouton and Bolles, 1979;
Rescorla, 1979; Wagner, 1981). Therefore, any observed Fos activation
during extinction may be involved in the inhibition, rather than
"unlearning," of the fearful behavior.
The role of Fos in aversive learning is supported by the results of ASO
blockade presented here. The blockade of Fos production during the
acquisition session diminished the subsequent expression of fearful
behavior, as well as increased the expression of nonfearful exploratory
behaviors. No acute effects of ASO on behavior were noted, decreasing
the likelihood that the ASO simply disrupted the acquisition of
conditioned fear. Additionally, no long-term changes in Fos expression
were noted when the ASO was given 72 hr before the acquisition session,
indicating that it is unlikely that any ASO activity was present during
the extinction session. Previous studies demonstrated that this Fos ASO
had no significant activity after 22-24 hr (Chiasson et al., 1992;
Hooper et al., 1994). Because the Fos protein is created several hours
after the initial stressful event, it is likely that Fos is
involved in the biochemical mechanism of consolidation of the fearful
memory, in agreement with the proposed role of Fos in learning and
memory (Dragunow, 1996). Alternatively, other explanations must be
considered. In this current study, state-dependent learning specific
for the ASO cannot be ruled out. This is not likely because (1)
previous learning studies with ASO directed against Fos protein have
not indicated state-dependent effects (Mileusnic et al., 1996) and, (2)
if state-dependent learning effects were present, they would have to be
specific for the ASO but not the sense oligonucleotide. The role of Fos
in the molecular mechanisms of learning and memory has been difficult
to validate. Previously, other researchers have not been able to
observe selective changes in learning because of changes in behavior
during training likely induced by the antisense blockade at that time
(Chiasson et al., 1992; Heilig et al., 1993; Hooper et al.,
1994; Moller et al., 1994; Hunter et al., 1995). We were able to
overcome this obstacle by (1) selecting an aversive task that involves
a select group of behaviors not normally expressed under controlled
conditions, (2) selecting a brain region thought to have a modulatory,
rather than primary, role in the expression of fearful behaviors, and
(3) using a task with a very short duration of acquisition (30 min).
The Fos activation in the mPFC demonstrated a selective regional
pattern appearing in the medial but not lateral (motor) prefrontal cortex. Anatomical studies of the subregions within the mPFC differ somewhat in the projection fields based on corticocortical afferents (van Eden et al., 1992). The dorsal mPFC, including the aCg, has been
demonstrated to have connections involving somatosensory and nonprimary
motor function (van Eden et al., 1992). The ventral mPFC, the PL and IL
together, seem to correspond to a limbic-related projection field
receiving input from perirhinal cortex areas, as well as direct and
indirect connections with limbic structures, such as the amygdala,
olfactory structures, nucleus accumbens, hypothalamus, and hippocampus
(Kita and Oomura, 1981; Swanson, 1981; Price and Slotnick, 1983;
Van Vulpen and Verwer, 1989; Sesack et al., 1989; Hurley et al.,
1991; van Eden et al., 1992). Together, the strong Fos activation to
the PL and IL seems to indicate a role for the limbic-related neuronal
system in the acquisition of aversive conditioning.
The role of the mPFC itself in conditioned fear is not clear but is
thought to be modulatory in nature. Lesions of the mPFC have resulted
in mixed results, including increasing (Morgan et al., 1993; Morgan and
LeDoux, 1995), decreasing (Frysztak and Neafsey, 1991),
and not altering (Gewirtz et al., 1997) the fear response. It
is likely that differences in these studies, such as the extent and
type of lesions, the training protocols, and other factors that could
alter the extent of conditioning or allow the neuronal adaptation to
the lesion, could contribute to this confusion. In our laboratory,
differences in the extent of excitotoxic lesions of the ventral mPFC
using two different lesioning protocols has resulted in divergent
effects on fear behaviors (our unpublished observations). For
these reasons, we have chosen less invasive approaches to investigate
the role of the mPFC in fear, including the approach presented in this
current report. Additionally, we have recently attempted to eliminate
the stress-induced increase in dopamine in the mPFC by dopaminergic
lesion (Morrow et al., 1999a) and by use of selective pharmacological
agents (Morrow et al., 1999b). These studies indicate that the mPFC has
a modulatory, rather than primary, role in the expression of fear.
We do not know the exact mechanism by which Fos in the mPFC is involved
in aversive learning. Electrophysiological studies have demonstrated
long-term potentiation and depression of excitatory potentials in
pyramidal cells in layer V of the prefrontal cortex (Hirsch and Crepel,
1990; Law-Tho et al., 1995). Such long-term changes, which can be
induced in seconds but remain for extended intervals (up to weeks),
have been proposed to be a mechanism of learning (Bliss and
Collingridge, 1993; Izquierdo, 1993). Dopamine, a neurotransmitter
activated by the conditioned fear protocol used in this report (Morrow
et al., 1995, 1996, 1997), has been demonstrated to induce long-term
depression in cells in layer V of the prefrontal cortex (Law-Tho et
al., 1994, 1995). It is possible that, through these changes in
excitability and subsequent secondary messenger systems, modified
transcription and translation of Fos and other immediate-early genes
occurs (Dragunow, 1996). This cascade in genetic activity presumably
leads to long-term alterations in the neuronal biochemistry or
structure which, in turn, are behaviorally expressed as, at least part
of, the acquisition of fear conditioning. This presumption is supported
by the behavioral results of ASO blockade of Fos-li presented here.
In conclusion, these studies report a selective activation of Fos-li in
the mPFC during the learning phase of a conditioned fear paradigm,
which we propose to be involved in the cellular mechanism of aversive
learning. From the effects of the blockade of the expression of Fos, we
conclude that Fos in the IL and PL maybe necessary for the
consolidation and subsequent expression of fearful memories.
| |
FOOTNOTES |
Received Feb. 16, 1998; revised April 12, 1999; accepted April 19, 1999.
This work was supported in part by National Institutes of Health Grants
MH-14092 and DA-11288. We thank Dr. Csaba Leranth and Marya
Shanabrough for their excellent technical advice and assistance.
Correspondence should be addressed to Dr. Bret A. Morrow, Department of
Pharmacology, Yale University School of Medicine, 333 Cedar Street, New
Haven, CT 06520-8066.
Dr. Inglis's present address: Department of Neurology, Yale University
School of Medicine, 333 Cedar Street, New Haven, CT 06520-8018.
| |
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ABSTRACT
INTRODUCTION
